JP2009519460A - Diagnosis and prognosis of colorectal cancer - Google Patents

Abstract

The present invention relates to a method for diagnosing colorectal cancer in a diagnostic sample of effective body tissue collected from a human subject, comprising detecting an increased concentration of protein in the diagnostic sample as compared to a control healthy human sample. Proteins include transforming growth factor β-inducible protein IG-H3 (SwissProt Acc. No. Q15582), SKP1 homolog allele G2 suppressor (isoform 2) (SwissProt Acc. No. Q9Y2Z0-2), hypothetical protein (URG4 (SwissProt Acc. No. Q9NWR7), calponin-2 (SwissProt Acc. No. Q99439), heat shock protein HSP90-β (SwissProt Acc. No. P08238), phosphoglycerate mutase 1 (SwissProt Acc. No. P18669), serpin C1 protein (SwissProt Acc. No. P01008), or haptoglobin precursor (SwissProt Acc. No. P00738). Alternatively, it comprises detecting a reduced concentration of protein in the diagnostic sample as compared to a control healthy human sample. Proteins include cellotransferrin (SwissProt Acc. No. P02787), 26S proteasome subunit p40.5 (SwissProt Acc. No.Q9UNM7), aldo-keto reductase family 1 member B10 (SwissProt Acc. No. O60218), fructosamine- 3-kinase (SwissProt Acc. No. Q9H479), peripherin (SwissProt Acc. No. P41219), α-2-macroglobulin (SwissProt Acc. No. P01023), serpin C1 protein (SwissProt Acc. No. P01008), or Apolipoprotein A IV (SwissProt Acc. No. P06727). The same protein can also be used for prognosis by detecting changes in protein concentration during colorectal cancer treatment.
[Selection] Figure 1

Description

  The present invention relates to the diagnosis and prognosis of colorectal cancer.

  Colorectal cancer (CRC) is the third most common cancer in the world. Carcinoembryonic antigen (CEA) (Gold and Freedman, 1965), cytokeratin (Moll et al., 1982), CA19-9, CA 242 (Nilsson et al., 1992), CA 72-4 (Fernandez -Fernandez et al., 1995), VEGF (Broll et al., 2001), p53 (Shiota et al., 2000; Hammel et al., 2000), HNP 1-3 (Albrethsen et al., 2005), RCAS1 (Yamagushi et al., 2005), apolipoprotein AI, apolipoprotein CI (Engwegen et al., 2006), complement C3a des-arg, α-1-antitrypsin, transferrin (Ward et al., 2006), PSME3 Serum tumor markers such as (Roessler et al., 2006) have been identified. However, there is a need to find new biomarkers for CRC in tumor tissue samples and body fluid (eg plasma) samples.

The present invention provides a method for the diagnosis of colorectal cancer in a diagnostic sample of useful body tissue taken from a human subject, which method comprises the following proteins in a diagnostic sample as compared to a healthy control human sample: Including detecting increased concentrations. Protein is
Transforming growth factor-β-inducing protein IG-H3 (SwissProt Acc. No. Q15582);
SKP1 homolog allele G2 suppressor (isoform 2) (SwissProt Acc. No. Q9Y2Z0-2);
Virtual protein (part of URG4) (SwissProt Acc. No. Q9NWR7);
Calponin-2 (SwissProt Acc. No. Q99439);
Heat shock protein HSP90-β (SwissProt Acc. No. P08238);
Phosphoglycerate mutase 1 (SwissProt Acc. No. P18669);
Serpin C1 protein (SwissProt Acc. No. P01008);
Or a haptoglobin precursor (SwissProt Acc. No. P00738).
Alternatively, it comprises detecting a reduced concentration of the following protein in a diagnostic sample as compared to a control healthy human sample. Protein is
Serotransferrin (SwissProt Acc. No. P02787);
26S proteasome subunit p40.5 (SwissProt Acc. No. Q9UNM7);
Aldo-keto reductase family 1 member B10 (SwissProt Acc. No. O60218);
Fructosamine-3-kinase (SwissProt Acc. No. Q9H479);
Peripherin (SwissProt Acc. No. P41219);
α-2-macroglobulin (SwissProt Acc. No. P01023);
Serpin C1 protein (SwissProt Acc. No. P01008);
Or apolipoprotein A IV (SwissProt Acc. No. P06727).

  Such proteins are referred to herein as “marker proteins” or “biomarkers”.

The same protein can also be used for prognosis by detecting concentration changes in the course of colorectal cancer treatment. Accordingly, the present invention also provides a method of monitoring the effect of colorectal cancer treatment in a subject. In this method, a change in the concentration of at least one protein in an effective body tissue sample collected from the subject at one stage of the treatment was collected from the subject prior to the treatment or at an early stage of the treatment. Detecting relative to the concentration of the protein in an effective body tissue sample. The protein is at least one of those specified above.

  Although the identification of the marker protein identified above is highly reliable, the present invention can instead be defined by the protein in a spot specifically expressed on a two-dimensional electrophoresis gel. That is, it can be defined by the protein identified in FIGS. 3 to 6 of the present specification regardless of the name or database ID.

<Definition>
The term “protein” (also referred to as “polypeptide”) is not limited to the sequences corresponding to the above accession numbers, but includes variants, mutants and isoforms thereof. A variant is defined as a naturally occurring mutation in the sequence of a polypeptide having high homology to a sequence and has substantially the same functional properties and immunological properties. A mutant is defined as an artificially created mutant. High homology is defined as homology of at least 90%, preferably at least 95%, and most preferably at least 99%. Variants may occur within a single species or may occur between different species. Polypeptide isoforms have the same function as polypeptides, but are encoded by different genes and may have slight differences in their sequences. Although the above proteins are derived from humans, the present invention also encompasses the use of corresponding polypeptides from other mammalian species.

  The term “specifically expressed” means that a stained protein-bearing spot is derived from a control sample or other comparative sample in a gel from a sample collected for diagnosis (“diagnostic sample”). It means to show higher or lower optical density than gel. Thus, the protein will be present at a higher or lower concentration in the diagnostic sample than in the control sample or other comparative sample.

  The term “control” refers to healthy tissue of a healthy subject, ie a subject not suffering from CRC, or the same human subject as the diagnostic sample.

  The term “increased / decreased concentration compared to control sample” does not imply that the comparison step is actually performed, because in many cases the concentration is abnormally high or low This is because it is obvious for an expert. In addition, when the CRC stage is observed gradually, or when the course of treatment is observed, relative to the concentration previously seen in the same subject at the beginning of disease progression, or at the beginning of treatment or before the start of treatment, A comparison can be performed.

  The term “binding partner” includes substances that recognize or have an affinity for a marker protein. This itself may or may not be labeled.

  The term “antibody” includes polyclonal antisera, monoclonal antibodies, fragments of antibodies such as single chain and Fab fragments, and recombinant antibodies. The antibody may be chimeric or may be a single species.

  The term “marker protein” or “biomarker” includes all biologically relevant forms of the identified protein. For example, a marker protein can exist in a body tissue in a glycosylated, phosphorylated, multimeric, or precursor form.

  As used herein, the term “diagnosis” includes determining the presence or absence of CRC, and also determining the stage at which CRC has progressed (or regressed during the course of treatment). This diagnosis may provide a prognostic basis for the patient's future outcomes.

  The term “effective body tissue” means any tissue in which a marker protein can reasonably be expected to accumulate in association with CRC. This may be a colorectal sample or a body fluid such as blood or a blood derivative (such as plasma or serum).

  The term “antibody array” or “antibody microarray” means an array of unique assignable elements on a continuous solid surface, thereby providing a distinct specificity for an antigen in each unique assignable element. The antibody possessed is then immobilized so that it can capture the target antigen and detect its degree of binding. Each unique assignable element is separated from all other unique assignable elements on the solid surface, and binding and detection of specific antigens interfere with any such unique assignable element adjacent There is no such thing.

  “Bead suspension array” means an aqueous suspension of one or more identifiable individual particles, whereby each particle contains code or fluorescent properties relating to its size and color, All beads with such a unique combination of coding properties are then covered with an antibody with a definite specificity for the antigen so that the target antigen can then be captured and the extent of its binding detected.

  A preferred diagnostic method comprises performing a marker protein binding assay. Any somewhat specific binding partner can be used. The binding partner is preferably labeled. The assay is preferably an immunoassay, in particular an immunoassay between a marker and an antibody that recognizes the protein (particularly a labeled antibody). The antibody may be prepared for a part or all of the antibody, and a monoclonal antibody or a polyclonal anti-human antiserum having high specificity for the marker protein is most preferable.

  Thus, the marker proteins described above are useful for the purpose of producing antibodies against them that can be used to detect increased or decreased concentrations of marker protein found in diagnostic samples. Such antibodies can be made by any of the methods well known in the immunodiagnostic field.

  The antibody can be against any biologically relevant state of the protein. Thus, for example, an antibody may contain a non-glycosylated form of a protein that exists in a glycosylated form in the body, a more mature form of a precursor protein (eg, a form without a signal sequence), or a related epitope of a marker protein. It can be prepared for a peptide possessed.

  The sample can be taken from any valid body tissue, in particular from the body fluid of a (human) subject, preferably blood, plasma or serum. Other available body fluids include cerebrospinal fluid (CSF), urine, and tears.

A preferred immunoassay is performed by measuring the extent of protein / antibody interaction. Any known immunoassay method can be used. A sandwich assay is preferred. In this method, a primary antibody against a marker protein is bound to a solid phase, such as a well of a plastic microtiter plate, and incubated with a sample and a labeled secondary antibody specific for the protein to be analyzed. Alternatively, an antibody capture assay can be used. Here, the sample sample may be bound to a solid phase, after which an anti-marker protein antibody is added and can be bound. After washing away unbound material, the amount of antibody bound to the solid phase is measured using an anti-primary antibody labeled secondary antibody.

  In another embodiment, a competition assay is performed between the sample and the labeled marker protein or a peptide derived therefrom. These two antigens compete for a limited amount of anti-marker protein antibody bound to the solid support. The labeled marker protein or peptide thereof can be preincubated with the antibody on the solid phase, whereby the marker protein in the sample replaces a portion of the marker protein or peptide thereof bound to the antibody.

  In yet another embodiment, the two antigens may compete in a single co-incubation with the antibody. After removing unbound antigen from the support by washing, the amount of label bound to the support is measured and the amount of protein in the sample is measured with reference to a pre-set standard titration curve.

  The label is preferably an enzyme. The enzyme substrate may be, for example, chromogenic, fluorescent, or chemiluminescent.

  The binding partner of the binding assay is preferably a labeled specific binding partner, but not necessarily an antibody. The binding partner is usually labeled itself, but may instead be detected by a secondary reaction in which a signal is generated, eg, from another labeled substrate.

  It is highly preferred to use an amplified form of the assay that produces an enhanced “signal” from a relatively low level of detection protein. One particular form of amplified immunoassay is the enhanced chemiluminescent assay. Conveniently, the antibody is labeled with horseradish peroxidase. It participates in a chemiluminescent reaction with luminol, a peroxide substrate, and a compound (typically 4-iodophenol or 4-hydroxycinnamic acid) that enhances the intensity and duration of the emitted light.

  Another preferred form of amplified immunoassay is immuno-PCR. In this technique, the antibody is covalently bound to any DNA molecule, including PCR primers, and the DNA to which the antibody is bound is amplified by the polymerase chain reaction. See E.R.Hendrickson et al., Nucleic Acids Research 23: 522-529 (1995). The signal is read as before.

  Alternatively, the diagnostic sample can be subjected to two-dimensional gel electrophoresis that results in a stained gel, with increased or decreased intensity of protein-containing spots on the stained gel, resulting in increased protein compared to the corresponding control or comparative gel A concentration or a decreasing concentration is detected. The associated spots and specific expression are listed in FIG. 2 as described below. The present invention includes such a method regardless of the identification of the marker protein described above and shown in FIG.

  In another embodiment, the diagnostic sample is a tissue section fixed by, for example, freezing or paraffin embedding and then subjected to immunohistochemistry.

  In a further embodiment, the diagnostic sample is subjected to in situ hybridization using a DNA probe against at least one messenger RNA of the proteins identified above.

In a still further embodiment, an increasing concentration of one or more of the following marker proteins overexpressed in CRC detects the increased level of autoantibodies relative to the level of autoantibodies in a control sample: Is detected. Autoantibody levels are determined by Western blot (from 1D or 2D electrophoresis) against their own tumor, autologous tumor, or CRC cell line, enzyme-linked immunosorbent assay (ELISA) using purified protein, protein microarray, or bead suspension. It can be detected by the array.
Transforming growth factor-β-inducing protein IG-H3 (SwissProt Acc. No. Q15582);
SKP1 homolog allele G2 suppressor (isoform 2) (SwissProt Acc. No. Q9Y2Z0-2);
Virtual protein (part of URG4) (SwissProt Acc. No. Q9NWR7);
Calponin-2 (SwissProt Acc. No. Q99439);
Heat shock protein HSP90-β (SwissProt Acc. No. P08238);
Phosphoglycerate mutase 1 (SwissProt Acc. No. P18669);
Serpin C1 protein (SwissProt Acc. No. P01008);
Haptoglobin precursor (SwissProt Acc. No. P00738)

  As an example, detection of autoantibodies against specifically increased proteins in patients with colorectal cancer can be performed as follows. Recombinant protein is expressed in baculovirus-infected insect cells and used to cover the surface of the microtiter plate. Serum / plasma collected from a patient suspected of having colorectal cancer is added to duplicate wells of each microtiter plate and incubated at 37 ° C. for 1 hour. Plates are aspirated, washed and incubated at 37 ° C. for 1 hour before adding anti-human IgG antiserum labeled with horseradish peroxidase (HRP). Finally, binding of anti-human antiserum is revealed by aspirating the plate, washing, and then adding tetra-methylbenzidine (TMB) that produces a colored product in the presence of HRP. The intensity of this colored product is measured by reading the plate at 450 nm. The same set of plates is tested unless the secondary antibody is HRP-labeled anti-human IgM antiserum. The levels of IgG and / or IgM autoantibodies against each CRC marker protein are elevated compared to the levels found in sera of healthy individuals who are not colorectal cancer.

  Diagnosis does not necessarily require a step of comparing protein concentration (or autoantibody concentration) with a control, but diagnosis can be performed with reference to either a control sample or a comparative sample. The present invention determines the stage of CRC, if necessary, with reference to previously obtained results from the same patient or with reference to standard values considered typical for the stage of the disease. Can be used for In this way, the present invention can be used to determine whether a disease has progressed, for example after treatment of a patient with a drug or candidate drug. The result can lead to a prognosis of disease outcome.

  The present invention further recognizes and binds to a marker protein represented by the specifically expressed two-dimensional gel electrophoresis spot identified above and / or shown in any of FIGS. Or using a partner substance with an affinity for it for diagnostic (and possibly prognostic) or therapeutic purposes. Thus, for example, antibodies against marker proteins that are appropriately humanized as needed may be used in therapy. The partner substance is usually an antibody and is used in any assay-compatible format, for example, conveniently immobilized as a bead or chip. The partner substance is either labeled or can interact with the label.

  The invention further comprises a kit for use in a diagnostic method comprising a partner substance as described above in an assay compatible format as described above for interaction with a protein present in a diagnostic sample.

  The present invention further comprises a kit for use in a diagnostic method comprising a specifically expressed protein as described above in an assay compatible format as described above for interaction with an autoantibody present in a diagnostic sample. .

  Diagnosis involves one, two, three or more marker proteins, or the specific expression of one, two, three, or more autoantibodies made against such proteins, or both Can be based on a combination. Furthermore, it can be part of a wider diagnosis where two or more different diseases are diagnosed. CRC is a method that involves detecting a change in another protein concentration in a diagnostic sample relative to a sample in a healthy human subject, and is used to detect at least one other disease (cancerous) in the same body tissue sample. Can be diagnosed with or without). These other disease (s) can be any that can be diagnosed in body tissue.

  Thus, in particular, in the present invention, an antibody chip or chip array capable of detecting one or more proteins that interact with an antibody, one or more autoantibodies that interact with a protein specifically expressed in colorectal cancer. It is contemplated to use a detectable protein chip or array of chips, a combination of both antibody and protein arrays.

  The following examples illustrate the invention.

<Example 1>
Fifteen patients were selected. From these patients, 15 colorectal samples corresponding to tumor tissue and 15 samples corresponding to adjacent healthy tissue were collected. A tumor tissue sample and a healthy tissue sample were placed on liquid nitrogen in a container and homogenized to a powdery hardness.

<2D gel electrophoresis (2DE) and mass spectrometry (MS)>
Prior to experimental studies, a 2DE separation optimization step was performed to cover the maximum number of proteins represented by the minimum number of 2D gels. Several IPG (immobilized pH gradient) strip ranges were tested (4-7, 3-7NL (NL = nonlinear), 3-10, 3-10NL). In the method of this study, the optimal range was 3-10 nonlinear. One analytical gel was provided for each sample (pH 3-10NL, 10% acrylamide, 100 μg protein loading). Gels were silver stained, scanned, and gel images were analyzed with ProGenesis software (v2005).

  The tumor tissue group was based on 13 analytical gels because the two tumor tissue samples had degraded. The healthy tissue group was based on 14 analytical gels because one gel was removed from the image analysis (spots smearing / diffuse). Two gel groups were compared with each other. The following criteria were used to select control spots: Spots are present at least within 60% of the gel, with a 2-fold down / up control, p-value <0.005.

  As a preparation gel, mix 4 healthy samples (healthy tissue mixture) and 4 tumor samples (tumor tissue mixture), and add 500 μg protein of each mixed sample to primary IPG (pH 3-10NL, 10% acrylamide) Loaded. The gel was scanned by Coomassie staining.

Collect the protein spot of interest from the preparation gel and use the Spot Handling Workstation (GE He
althcare). Peptide profiles are created with MALDI-ToF (Matrix Assisted Laser Desorption / Ionization-Time-of-Flight Mass Spectrometer), analyzed with Ms-Fit software (Protein Prospector), and searched with IPI (International Protein Index) database did.

[result]
<Image analysis method and number of target control protein spots>
Gel images were analyzed with ProGenesis (v2005). Spot detection, matching, background removal, and normalization were done automatically by ProGenesis. The background removal method used a proprietary background removal algorithm established by Nonlinear Dynamics. This algorithm calculated the background level based on the entire gel surface model. The normalization mode used was the “Total Spot Volume” method. In this method, the volume of each spot in the gel is divided by the total volume of all spots. Since this method tends to produce very small values, the result was multiplied by a factor. For example, if the default value of 100 is used for the coefficient, a spot percentage volume is obtained. In this study, we decided to multiply an integer larger than the default value specified by ProGenesis by a factor of 1000 that can be used for data analysis. Then export the spot data to Excel and use macros to control the coefficient of variation (CV,%), Student T test, Mann-Whitney test, and the tumor volume to healthy group spot volume ratio (Regulation
factor) was calculated within a confidence level of 99.5 (p <0.005).

  Figure 1 shows the number of spots detected on each gel, the sum of all spot volumes (IOD), and the magnification -SF- (the sum of the volumes of all spots in the reference gel, the sum of the volumes of all spots in the gel. Divided). In FIG. 1, for each gel, the number of spots detected, the total volume of all spots (IOD), and the multiple (S.F.) are shown. Gel n_03-0562_2217 corresponds to the reference gel. In this table, it can be seen that all gels have a magnification (0.6 <S.F. <1.5) exceeding the experimental value. These 27 gels were reflected in the image analysis.

  In this study, these selection criteria were applied based on experience. Spots should be seen at least within 60% of the gel, with a 2-fold up / down change of 99.5 confidence (Student t test and / or Mann-Whitney test; p <0.005). Approximately 900 spots were subjected to statistical analysis due to the requirement that spots exist within 60% of the gel. It was found that 31 spots were expressed specifically between tumor tissue and healthy tissue. These spots are listed in FIG. Eighteen spots were up-regulated or predominant in the tumor tissue group, and 13 spots were down-regulated or predominant in the healthy tissue group. The gel spot positions are shown in FIGS. Scatter plots of normalized volume from selected spots are shown in FIGS.

  The 99.5 confidence level is a very narrow interval, meaning high accuracy, and therefore represents a very strict standard. Indeed, a p-value of 0.005 means that this result can occur with a probability of less than 1 per 200, in other words, it can be expected that one spot will be randomly selected from 200 selected spots. . In the case of this study, about 5 to 900 spots could be randomly selected with a confidence of 99.5.

All statistical analyzes of normalized data must be accompanied by a negative control analysis of the data. One of these negative controls is a pseudo-group analysis, ie analysis by mixing data columns. For example, if an experiment is composed of six control gels and experimental gels, the two groups to be compared must each contain three control gels plus three experimental gels (pseudogroups). This “pseudo-group” comparison shows (i) how many spots can be randomly selected by ignoring the potential noise of the dataset in the analysis, and (ii
) Ensure that a solid control spot is selected with true statistical significance from actual comparisons. By performing this type of test, it becomes clear what kind of selection criteria can reveal the true changes within the experimental group. If, after performing a “pseudo-group” comparison, the same number of changes as the actual comparison is found, then the data set is “empty” in the group-specific changes.

  Therefore, in this study, two pseudogroups were created by mixing half of healthy tissue gel and half of tumor tissue gel, and pseudogroup 1 and pseudogroup 2 were compared. Pseudogroup analysis includes all gels that were included in the actual comparison. The same spot selection method (within 60% of the gel, p <0.005 and 2-fold change) was used for this pseudogroup analysis. As shown in the table of FIG. 7, five spots corresponding to the theoretical values calculated above were selected.

<Mass spectrometric identification of target protein spot>
The prepared healthy tissue gel and the prepared tumor tissue gel were subjected to experiments together with a mixture of four healthy tissue samples and a mixture of four tumor tissue samples, respectively. These gels were stained with Coomassie blue, target spots were collected and processed in a Spot Handling workstation (GE Healthcare), and the peptides were loaded onto a MALDI-ToF target. MS spectra were analyzed with Ms-Fit software and IPI database. As shown in Table 2, the total protein spots presented were successfully identified with an average of 13 peptides per protein with a protein identity of approximately 34% and a measurement error of 4.7 ppm. 31 protein spots correspond to 22 different protein species.

[Discussion]
<Verification of used technology>
This study revealed that a protein specifically expressed between tumor tissue and healthy tissue was identified by 2D gel electrophoresis. Several proteomic studies have already been performed to find CRC biomarkers (Albrethsen et al., 2005; Mori et al., 2005; Ahmed, 2005; Alessandro et al., 2005; Drew et al. , 2005, Alfonso et al., 2005; Friedman et al., 2004; Lawrie LC et al., 2001). Some of the proteins found to be regulated have already been identified. : Glycyl-tRNA synthetase (spot 241), 60 kDa heat shock protein (spots 543 and 543 left), adenosyl homocysteinase (spot 768), inorganic pyrophosphatase (spot 1015), annexin A4 (spot 1049), F-actin capping protein β subunit (spot 1052), tropomyosin alpha 4 (1081 and 1458), Rho GDP dissociation inhibitor 1 (spot 1171), 14-3-3 protein α / β (spot 1171), translationally controlled tumor protein (Spot 1229) and serum albumin (spots 733, 346, 364, 404, 435, 460, 462, 519, 877, 1060 and 1256). The controls observed with all proteins except serum albumin spot 733 were consistent with those found previously. The fact that these proteins were identified in this study indicates that the samples analyzed and the accuracy and sensitivity of the method used are high. On the other hand, these observations confirm the high resolution of the 2-DE protocol in this study. Indeed, all publications dealing with 2D electrophoresis (Mori et al., 2005; Alfonso et al., 2005; Friedman et al.
al., 2004) used DIGE (fluorescence labeled 2-D difference gel electrophoresis) technology (GE Healthcare).

<New CRC biomarker>
This study identified proteins in colon cancer tissues: transforming growth factor-beta-inducible protein IG-H3 (spot 407), SKP1 homolog allele G2 suppressor (spot 949), hypothetical protein (spot 975), calponin- 2 (spot 983), heat shock protein HSP90-β (spot 1191), cellotransferrin (spot 361), 26S proteasome subunit p40.5 (spot 877), aldo-keto reductase family 1 member B10 (spot 943), Fructosamine-3-kinase (spot 943), peripherin (spot 543) and phosphoglycerate mutase 1 (spot 1354).

  This study was conducted to (i) find new biomarkers for CRC, and (ii) higher expressed proteins are easier and more accurate to detect than lower expressed proteins in cancer stages. The study presents here information on the function of proteins up-regulated in cancer tissues.

  The transforming growth factor-β-inducing protein IG-H3 (spot 407) is a polypeptide of unknown function and is expressed in corneal keratinocytes (Escribano et al., 1994). The coding gene TGFB1 has been shown to be significantly elevated in colorectal cancer and adenoma (Zhang et al., 1997; Buckhaults et al., 2001).

  The SKP1 homolog allele G2 suppressor (spot 949) may be involved in ubiquitination and proteasome degradation of the target protein. Two isoforms have been confirmed by immunoblotting (Niikura and Kitagawa, 2003).

  The hypothetical protein (spot 975) has no function, but its amino acid sequence closely matches the C-terminal sequence of the URG4 protein (611-922) as shown in FIG. Overexpression of URG4 in HepG2 cells accelerated tumor growth in nude mice (Tufan et al., 2002).

  Calponin-2 (spot 983) is a protein associated with thin filaments that is involved in the control and regulation of smooth muscle contraction. It can bind to actin, calmodulin, troponin C and tropomyosin. Calponin-actin interaction inhibits actomyosin Mg-ATPase activity (Kitching et al .. 2002).

  The heat shock protein HSP90-β (spot 1191) is a molecular chaperone that is constitutively expressed and induces normal folding, intracellular kinetics, and many proteolytic turnovers of key regulators of cell growth and survival . The intrinsic guard duty of the heat shock protein HSP90 appears to provide a very promising unique anticancer method (Whitesell et al., 2005).

  Phosphoglycerate mutase 1 (spot 1354) is a glycolytic enzyme. Recently, a small molecule, MJE3, that suppresses breast cancer cell growth has been identified (Evans et al., 2005). MJE3 binds to phosphoglycerate mutase 1 and results in enzyme inhibition. The authors suggest that cancer cells depend on glycolysis for viability and have listed phosphoglycerate mutase 1 as a future therapeutic target.

[Conclusion]
The purpose of this study was to screen tissue and blood samples from patients with CRC to find new biomarkers. In this study, 2-DE gel electrophoresis was used to display proteins, silver staining was used to detect protein spots, and mass spectrometry was used to identify proteins of interest.

In this study, the result of the tissue analysis was shown. Sample preparation was performed by skilled technicians using a unique and identical protocol for all samples. All samples were processed simultaneously. The samples were observed to be different colors (approximately red / pink / brown). 2D gels from two tumor samples showed proteolysis and showed anomalous sample profile characteristics (no protein spots in high molecular weight gel region, increased number of protein spots in low molecular weight gel region, gel The electrophoresis tip became darker at the bottom. These two samples not only had the lowest protein concentration, but were also difficult to make into a powdery hardness.

  In addition, colon cells were perfused with blood, and it was clear that albumin represented ˜50% of protein content. Since the sample colors are different (pink / red / brown), it can be imagined that the samples contained different amounts of albumin. This may explain why albumin was identified as a regulatory protein. Sample preparation is known to be an important step and precautions should be taken before freezing the sample. For example, it is suggested to place the tissue on paper and leave it for a few minutes to make sure that blood is aspirated from the tissue. This kind of precaution can prevent misinterpretation of observations.

  In the 2D method of this study, it was found that 31 protein spots were expressed specifically between healthy tissue and tumor tissue. All of these spots were identified by peptide mass fingerprinting (PMF), which corresponded to 22 different protein species.

  Interestingly, the 11 proteins identified (50%) corresponded to new markers for CRC. The six proteins are of particular interest because they were overexpressed in tumor tissue. Eleven other proteins identified (50%) have already been published from biomarker discovery studies using fluorescent dyes (DIGE technology). These eleven proteins may be considered as verifying the capabilities of the method of this study and represent the high sensitivity of the silver staining protocol of this study. The fact that these proteins were rediscovered in this study using different sample sets confirms the usefulness of these markers.

<Example 2>
Two-dimensional gel electrophoresis and mass spectrometry were used to analyze protein expression in tumor and control plasma samples. Each sample was removed individually and separated on a 2DE gel. A large difference was seen for seven spots with statistically significant changes between the tumor sample and the control sample (at least spots within 60% of the gel, with 1.5-fold down / up-regulation, And p-value <0.005). All protein spots were identified by MALDI-ToF MS, which corresponded to four different protein species.

  15 colorectal cancer patients and 20 healthy controls were selected. All samples were removed simultaneously on an Agilent MARS column and proteins were TCA precipitated.

<2D gel electrophoresis, image analysis, mass spectrometry>
One analytical gel was provided for each plasma sample (pH 3-10NL, 10% acrylamide, 100 μg protein loading). Gels were silver stained, scanned, and gel images were analyzed with ProGenesis software (v2006).

  All 45 analysis gel images (20 healthy plasma gels as control group and 15 tumor plasma gels as tumor group) were incorporated into the image analysis. The two groups were compared with each other. The following criteria were used to select control spots. Spots are at least within 60% of the gel, with a 1.5-fold change, and a p-value <0.005 (Mann-Whitney test).

  The prepared gel was mixed with 5 healthy samples and 5 tumor samples, and each mixed sample 350 μg protein was loaded onto the primary IPG (pH 3-10NL, 10% acrylamide). The gel was Coomassie stained and scanned.

The protein spots of interest were collected from the prepared gel and processed with a Spot Handling Workstation (GE Healthcare). Peptide profiles were generated with MALDI-ToF, analyzed with Ms-Fit software (Protein Prospector), and searched in the IPI database. Where cross-references with SwissProt are available in the IPI database, reference numbers in SwissProt are indicated (Figure 19).

[result]
<1-Image analysis method and the number of detected control protein spots>
Gel images were analyzed with ProGenesis (v2006). Spot detection, matching, background removal and normalization were done automatically by ProGenesis. The background removal method used was a patented background removal algorithm established by Nonlinear Dynamics. Based on the entire gel surface model, the algorithm calculated the background. As the normalization mode, the “total spot volume” method was used. In this method, the volume of each spot in the gel is divided by the total volume of all spots. Since this method tends to produce very small values, the result was multiplied by a factor. For example, using the default value of 100 as the factor results in spot percent volume.

  In this study, we decided to multiply an integer larger than the default value specified by ProGenesis by a factor of 1000 that can be used for data analysis. The spot data is then exported to Excel, and using macros, the coefficient of variation (CV,%), Student T test, Mann-Whitney test, and the control factor between the tumor volume and the healthy group spot volume ratio (Regulation factor) Was calculated within a confidence of 99.5 (p <0.005).

  In this study, we applied these selection criteria based on experience. Spots should be seen at least within 60% of the gel and should have a 1.5-fold up / down change within 99.5 confidence (Mann-Whitney test; p <0.005). Approximately 650 spots were subjected to statistical analysis, with the requirement that spots exist within 60% of the gel. Seven spots were found to be specifically expressed between tumor plasma samples and healthy plasma samples. These spots are listed in FIG. Two spots were up-regulated in the tumor sample group and five spots were down-regulated in the healthy sample group. The spot position in the gel is shown in FIG.

<2- Mass spectrometric identification of target protein spots>
The prepared healthy plasma gel and the prepared tumor plasma gel were subjected to experiments together with a mixture of five healthy samples and a mixture of five tumor samples, respectively. These gels were stained with Coomassie blue, the target protein was collected and processed with a Spot Handling Workstation (GE Healthcare), and the peptide was loaded on a MALDI-ToF target. MS spectra were analyzed with Ms-Fit software and IPI database. As shown in FIG. 19, all presented protein spots were successfully identified with an average of 13 peptides per protein and a protein coverage of approximately 20%. Seven protein spots corresponded to four different protein species (FIG. 19).

[Discussion]
<1- All proteins that were previously known to be regulated in tissues were found not to be regulated in plasma at all>
In Example 1 based on the analysis of specific protein expression between tumor tissue and healthy tissue, 22 specific regulatory proteins were identified. Despite the fact that some of these 22 proteins were already found in plasma, none of them was found to be regulated in plasma.

The fact that two different sets of control proteins have been identified, one marker set in CRC tissue samples and one marker set in plasma samples, is directly related to the metabolic characteristics of cancer cells in tissues. It suggests that it is not transferred to the current. This may be due to the role of the cell membrane filter of tissue cells and / or the high dilution rate into the blood that cannot be corrected by the 2D gel method. At least in experiments in tissues, serum albumin has been identified as a control protein. This protein was removed from the plasma (see experimental procedure above), and thus was not found in the plasma samples examined.

<Regulatory protein detected in 2-CRC plasma>
In plasma experiments, two up-regulated spots were found in tumor samples (spots 748; 2049), identified as serpin C1 protein and haptoglobin precursor, respectively. There are also five down-regulated spots corresponding to three different protein species α-2-macroglobulin (spots 707; 710; 711), serpin C1 protein (spot 1040) and apolipoprotein A IV (spot 1149). Found in tumor plasma (Figure 19).

  Serpin C1 protein (or antithrombin 3) is a very important protease inhibitor in plasma that controls the blood coagulation cascade. Recently, Sierko and coll. (2006) observed that in many cases of colon cancer, the expression of antithrombin 3 has very low intensity in some cancer foci. In about 15% of cases, there was no expression of AT3, but in another 15% of experimental colon cancer fragments, AT3 was easily detected. In the gel of this study, two spots were identified as antithrombin 3, one spot was down-regulated (1040) and the other was up-regulated (748). Different control of isoforms of the same protein in colorectal cancer serum samples has already been observed. In fact, Rodriguez-Pineiro and coll. (2006) found one clusterin N-glycosylated isoform that was up-regulated in the CRC sample and 15 clusterin N-glycosylation down-regulated or absent in the CRC sample. An isoform has been identified. Since the four known sites of antithrombin N-glycosylation are represented by Swiss-Prot (P01008), it can be imagined that a similar event may have occurred in antithrombin 3, which explains the observations of this study.

  Haptoglobin protein has two chains α and β. The main function of haptoglobin is to bind hemoglobin (Hb) to form a stable Hp-Hb complex, thereby preventing Hb-induced oxidative tissue damage. This protein has been shown to be regulated in Alzheimer's disease samples. Spots identified in this study have already been identified as haptoglobin by another group (see note, spots shown in red), confirming the identification of this study.

  Bresalier and coll. (2004) identified that the haptoglobin protein is a ligand for galectin-3. Galectin-3 is a β-galactoside-binding protein involved in tumor progression and colorectal cancer metastasis. They concluded that the main circulating ligand of galectin-3, elevated in the serum of colon cancer patients, is the cancer-associated glycosylated form of haptoglobin. Immunohistochemical staining confirmed the absence of haptoglobin in the healthy colon and the ectopic expression of haptoglobin in colon cancer and adenomatous polyps.

The α-2-macroglobulin protein can inhibit all four classes of proteinases by a unique “capture” mechanism. Along with myeloperoxidase, α-2-macroglobulin is involved in a molecular pathway that leads to β-amyloid deposition (Du et al., 1998). α-2-macroglobulin can bind to prostate specific protein (PSA), after which the PSA-A2M complex is not detected by conventional PSA immunoassays (Lilja et al., 1991). α-2-Macroglobulin has been identified as a marker for hepatocellular carcinogenesis (Kawakami et al., 2005) but is not a marker for colorectal cancer. This protein is also known to be down-regulated in Alzheimer's disease samples. The mature protein has a molecular weight of 160796 Da, which is inconsistent with the observed molecular weight of spots in the gel (˜100 kDa, FIG. 20, spots 707; 710; 711). It can be imagined that the proteins identified in this study can correspond to fragments of the full-length sequence of α-2-macroglobulin. It can be imagined that the spots identified as α-2-macroglobulin belong to the same spot chain (FIG. 20) and that the difference between these spots may be due to post-translational modification of the protein.

Apolipoprotein A4 is a major component of HDL (high density lipoprotein) and chylomicron and is required for the activation of lipoprotein lipase by apolipoprotein C2. This protein is produced in the intestine and then secreted into the plasma. In this study, apolipoprotein A4 expression was suppressed in CRC patients, corresponding to the opposite expression observed in hepatocellular carcinoma (Kawakami et al., 2005) and pancreatic cancer (Zervos et al., 2006). To do.

[Conclusion]
The purpose of this study was to screen removed plasma samples from patients with CRC to find new biomarkers. Proteins were displayed using 2-DE gel electrophoresis, protein spots were detected using silver staining, and the target protein was identified using mass spectrometry.

  Plasma removal, sample preparation, and 2D gel electrophoresis were performed by skilled technicians. All samples were processed simultaneously. Since the sample was fully packaged and received in good condition, no problems occurred during these critical steps and no degradation was observed in the 2D gel.

  It was found that seven protein spots were specifically expressed between the plasma of healthy subjects and tumor patients. All these spots were identified by PMF and corresponded to four different protein species.

  Interestingly, one identified protein (haptoglobin) is already known to be associated with CRC. This may support the validity of the method of this study. The identification of the same colorectal cancer marker in different studies and groups reinforces its validity.

  Furthermore, proteins that were selectively expressed in healthy patients (α-2-macroglobulin and apolipoprotein A IV) can be considered surrogate markers. These markers can be measured after therapy or surgical procedure. If the protein is newly expressed in the patient, it may indicate a successful treatment or treatment. In order to support the preliminary results of this study, further studies should be performed on a larger set of samples.

[References]
Ahmed FE, Expert Rev Mol Diagn 2005; 5: 353-375.
Albrethsen J, Bogebo R, et al, BMC Cancer 2005; 5: 8-17.
Alessandro R, Belluco C, et al, Clin Colorectal Cancer 2005: 4: 396-402.
Alfonso P, Nunez A, et al, Proteomics 2005; 5: 2602-2611.
Bresalier RS, Byrd JC, Tessler D, et al, Gastroenterology, 2004; 127: 741-748.
Broll R, Erdmann H, et al, Eur. J Surg Oncol 2001; 27: 37-42.
Buckaults P, Rago C, et al, Cancer Res 2001; 61: 6996-7001.
Drew JE, Rucklidge GJ, et al, Biochem Biophys Res Commun 2005; 330: 81-87.
Du Y, Bales KR, Dodel RC, et al, J Neurochem. 1998; 70: 1182-1188.
Enwegen J., Helgason H., Cats A., et al, World J. Gastroenterology 2006; 12: 1536-1544.
Escribano J, Hernando N, et al, J Cell Physiol 1994; 160: 511-521.
Evans MJ, Saghatelian A, et al, Net Biotechnol 2005; 23: 1303-1307.
Fernandez-Fernandez L, Tejero E, et al, Eur. J Surg Oncol 1995; 21: 388-390.
Friedman D, Hill S, et al, Proteomics 2004; 4: 793-811.
Gold P, Freedman SO, J. Exp. Med. 1965; 121: 439-162.
Hammel P, Soussi T, Rev Med Interne 2000; 21: 167-173.
Kawakami T., Hoshida Y., Kanai F., et al, Proteomics, 2005; 5; 4287-4295.
Kitching R, Qi S, et al, J Bone Miner Metab 2002; 20: 269-280.
Lawrie LC, Curran S, et al, J Clin Pathol: MolPathol 2001; 54: 253-258.
Lilja H, Christensson A, Dahlen U, et al, Clin Chem 1991; 37: 1618-1625.
Moll R, Franke WW, et al, Cell 1982; 31: 11-14.
Mori Y, Kondo T, et al, J Chromatogr B Analyt Technol Biomed Life Sci. 2005; 823: 82-97.
Niikura Y, Kitagama K, DNA Seq 2003; 14: 436-441.
Nilsson O, Johannsson C, et al, Br J Cancer 1992; 65: 215-221.
Rodriguez-Pineiro AN, Paez de la Cadena M, Lopez-Saco A, et al, Mol.Cell.
Proteomics, 2006; in press.
Roessler M, Rollinger W, Mantovani-Endl L, et al, Mol. Cell. Proteomics 2006; in
press.
Sierko E, Zawadzki RJ, Zimnoch L, Pol. Merkuriusz Lek., 2006; 20; 462-467.
Shiota G, Ishida M, et al, Dig Dis Sci 2000; 45: 122-128.
Tufan NL, Lian Z, et al, Neoplasia 2002; 4: 355-368.
Ward DG, Suggett N, Cheng Y, et al, British J. Cancer 2006; 6: 1898-1905.
Whitesell L, Lindquist SL, Nature Rev Cancer 2005; 5: 761-772.
Yamagushi K, Enjoji M, et al, World J Gastroenterol 2005; 11: 5199-5202.
Zervos EE, Tanner SM, Osborne DA, et al, J. Surg. Res. 2006: in press.
Zhang L, Zhou W, et al, Science 1997; 276: 1268-1272.

It is a table | surface of the result of the following Example 1, and shows the number of the spots detected with respect to each gel, the sum total of the volume of all the spots, and magnification. It is a table | surface of the result of the following Example 1, and shows the protein detected by each spot. 2 is a photograph of a two-dimensional gel of protein extracted from tumor tissue by the method described in Example 1. FIG. The pattern shown corresponds to a 2-D PAGE image of a silver stained gel. The molecular weight marker in kilodaltons (kDa) is shown on the ordinate, and the isoelectric point (pI) is shown on the abscissa, increasing from left to right. The spot number indicates the protein spot up-regulated in the tumor tissue. It is the photograph of the same gel, and a spot number shows the spot of the protein which occupies most in tumor tissue. Similar gel pictures, spot numbers indicate protein spots down-regulated in tumor tissue. It is the photograph of the gel corresponding to the protein extracted from the healthy tissue, and a spot number shows the spot of the protein which occupies most in a healthy tissue. FIG. 4 is a table showing the number of spots randomly selected by pseudogroup analysis as described in Example 1 below. BLAST sequence of URG4 protein of hypothetical protein (SeqA) is shown. The amino acid sequence of the hypothetical protein (1-312) perfectly matches the C-terminal amino acid sequence of the URG4 protein (611-922). FIG. 6 is a scatter plot showing the normalized volume from spot 361 identified as cellotransferrin. “Control” refers to the normalized volume intensity from spots detected on the control sample gel, and “Test” refers to the normalized volume intensity from spots detected on the tumor sample gel. FIG. 6 is a corresponding scatter plot showing the normalized volume from spot 407 identified as transforming growth factor-β-inducing protein IG-H3. FIG. 5 is a corresponding scatter plot showing the normalized volume from spot 543 identified as peripherin. FIG. 6 is a corresponding scatter plot showing the normalized volume from spot 877, identified as a mixture of two proteins, serum albumin and 26S proteasome subunit p40.5. FIG. 10 is a corresponding scatter plot showing the normalized volume from spot 943, identified as a mixture of two proteins, aldo-keto reductase family 1 member B10 and fructosamine-3-kinase. FIG. 6 is a corresponding scatter plot showing normalized volume from spot 949 identified as suppressor of SKP1 homolog allele G2 (isoform 2). FIG. 10 is a corresponding scatter plot showing the normalized volume from spot 975 identified as a virtual protein (part of URG4). FIG. 10 is a corresponding scatter plot showing the normalized volume from spot 983 identified as calponin-2. A corresponding scatter plot showing the normalized volume from spot 1191 identified as heat shock protein HSP90-β is shown. A corresponding scatter plot showing the normalized volume from spot 1354 identified as phosphoglycerate mutase 1 is shown. It is a table | surface of the result of the following Example 2, and shows the protein detected by each spot. 2 is a photograph of a two-dimensional gel of protein extracted from a plasma sample by the method described in Example 2. 2 is a scatter plot of spot 707 identified as α-2-macroglobulin described in Example 2. FIG. FIG. 6 is a corresponding scatter plot of spot 710 identified as α-2-macroglobulin. FIG. 6 is a corresponding scatter plot of spot 711 identified as α-2-macroglobulin. FIG. 10 is a corresponding scatter plot of spot 1040 identified as serpin C1 protein (antithrombin 3). FIG. 4 is a corresponding scatter plot of spot 1149 identified as apolipoprotein A-IV. FIG. 7 is a corresponding scatter plot of spot 748 identified as serpin C1 protein (antithrombin 3). FIG. 10 is a corresponding scatter plot of spot 2094 identified as a haptoglobin precursor.

  9 to 18 and 21 to 27, the result of “Control” is shown on the left side of the result of “Test”.

Claims (35)

A method for diagnosing colorectal cancer in a diagnostic sample of effective body tissue collected from a human subject, comprising the step of detecting an increased concentration of protein in the diagnostic sample as compared to a control healthy human sample, Protein is
Transforming growth factor-β-inducing protein IG-H3 (SwissProt Acc. No. Q15582);
SKP1 homolog allele G2 suppressor (isoform 2) (SwissProt Acc. No. Q9Y2Z0-2);
Virtual protein (part of URG4) (SwissProt Acc. No. Q9NWR7);
Calponin-2 (SwissProt Acc. No. Q99439);
Heat shock protein HSP90-β (SwissProt Acc. No. P08238);
Phosphoglycerate mutase 1 (SwissProt Acc. No. P18669);
Serpin C1 protein (SwissProt Acc. No. P01008);
Or a haptoglobin precursor (SwissProt Acc. No. P00738),
Alternatively, detecting a reduced concentration of the protein in the diagnostic sample as compared to a control healthy human sample, the protein comprising:
Serotransferrin (SwissProt Acc. No. P02787);
26S proteasome subunit p40.5 (SwissProt Acc. No. Q9UNM7);
Aldo-keto reductase family 1 member B10 (SwissProt Acc. No. O60218);
Fructosamine-3-kinase (SwissProt Acc. No. Q9H479);
Peripherin (SwissProt Acc. No. P41219);
α-2-macroglobulin (SwissProt Acc. No. P01023);
Serpin C1 protein (SwissProt Acc. No. P01008);
Or Apolipoprotein A IV (SwissProt Acc. No. P06727).   The method according to claim 1, characterized in that the protein concentration in the diagnostic sample is increased or decreased by at least 2-fold compared to the control sample.   The method according to claim 1 or 2, wherein the detection is performed on the diagnostic sample by the protein binding assay.   4. The method of claim 3, wherein the binding assay comprises interacting the protein of the diagnostic sample with a specific binding partner and detecting the interaction.   5. A method according to claim 4, characterized in that the specific binding partner is labeled.   6. The method according to claim 4 or 5, characterized in that the specific binding partner is an antibody or antibody fragment that recognizes the protein.   7. The method according to any one of claims 1 to 6, characterized in that the control sample is taken from healthy tissue of the same human subject as the diagnostic sample.   7. A method according to any one of the preceding claims, characterized in that the control sample is taken from a healthy tissue of a human subject different from that which provides the diagnostic sample.   The method according to claim 8, wherein the body tissue is a body fluid, and the body fluid is subjected to an immunoassay.   10. The method of claim 9, wherein the immunoassay is a Western blot, enzyme linked immunosorbent assay (ELISA), antibody microarray, or bead suspension array.   9. The diagnostic sample according to any one of claims 1 to 8, wherein the diagnostic sample is a frozen or paraffin-embedded tissue section, and the tissue section is subjected to immunohistochemical staining. Method.   The diagnostic sample is subjected to two-dimensional gel electrophoresis to yield a stained gel, and the increased or decreased concentration of the protein is increased in intensity of protein-containing spots on the stained gel compared to the corresponding control gel 3. A method according to claim 1 or 2, characterized in that it is detected by decreasing intensity.   The increased intensity of spots 407, 949, 975, 983, 1191 or 1354 or the decreased intensity of spots 361, 877, 943 or 543 in FIGS. 3 to 6 is detected. The method described.   The method according to claim 1 or 2, comprising in situ hybridization using a DNA probe against at least one messenger RNA of the proteins identified in claim 1. An increasing concentration of one or more of the following marker proteins is detected by detecting an elevated level of autoantibodies relative to the level of autoantibodies in the control sample: The method according to claim 1 or 2.
Transforming growth factor-β-inducing protein IG-H3 (SwissProt Acc. No. Q15582);
SKP1 homolog allele G2 suppressor (isoform 2) (SwissProt Acc. No. Q9Y2Z0-2);
Virtual protein (part of URG4) (SwissProt Acc. No. Q9NWR7);
Calponin-2 (SwissProt Acc. No. Q99439);
Heat shock protein HSP90-β (SwissProt Acc. No. P08238);
Phosphoglycerate mutase 1 (SwissProt Acc. No. P18669);
Serpin C1 protein (SwissProt Acc. No. P01008);
Or haptoglobin precursor (SwissProt Acc. No. P00738)   16. The method according to claim 15, characterized in that the level of autoantibodies is detected by Western blot, ELISA, protein microarray or bead suspension array.   15. A method according to any one of the preceding claims, characterized in that an increase or decrease in the concentration of one or more marker proteins identified in claim 1 is detected.   15. A method according to any one of claims 1-8 and 11-14, characterized in that the effective body tissue is a colorectal sample. A method for monitoring the effect of treatment of colorectal cancer in a subject, wherein a change in the concentration of at least one protein in an effective body tissue sample taken from the subject during the treatment phase is measured before or after the treatment. Detecting compared to the concentration of the protein in an effective body tissue sample taken from the subject at an early stage of the treatment, the protein comprising:
Transforming growth factor-β-inducing protein IG-H3 (SwissProt Acc. No. Q15582);
SKP1 homolog allele G2 suppressor (isoform 2) (SwissProt Acc. No. Q9Y2Z0-2);
Virtual protein (part of URG4) (SwissProt Acc. No. Q9NWR7);
Calponin-2 (SwissProt Acc. No. Q99439);
Heat shock protein HSP90-β (SwissProt Acc. No. P08238);
Phosphoglycerate mutase 1 (SwissProt Acc. No. P18669);
Serpin C1 protein (SwissProt Acc. No. P01008);
Haptoglobin precursor (SwissProt Acc. No. P00738):
Serotransferrin (SwissProt Acc. No. P02787);
26S proteasome subunit p40.5 (SwissProt Acc. No. Q9UNM7);
Aldo-keto reductase family 1 member B10 (SwissProt Acc. No. O60218);
Fructosamine-3-kinase (SwissProt Acc. No. Q9H479);
Peripherin (SwissProt Acc. No. P41219);
α-2-macroglobulin (SwissProt Acc. No. P01023);
Or Apolipoprotein A IV (SwissProt Acc. No. P06727). The change in protein concentration is a decrease, and the protein is
Transforming growth factor-β-inducing protein IG-H3 (SwissProt Acc. No. Q15582);
SKP1 homolog allele G2 suppressor (isoform 2) (SwissProt Acc. No. Q9Y2Z0-2);
Virtual protein (part of URG4) (SwissProt Acc. No. Q9NWR7);
Calponin-2 (SwissProt Acc. No. Q99439);
Heat shock protein HSP90-β (SwissProt Acc. No. P08238);
Phosphoglycerate mutase 1 (SwissProt Acc. No. P18669);
Serpin C1 protein (SwissProt Acc. No. P01008);
Or haptoglobin precursor (SwissProt Acc. No. P00738)
The method according to claim 19, wherein: The change in protein concentration is increased, and the protein is
Serotransferrin (SwissProt Acc. No. P02787);
26S proteasome subunit p40.5 (SwissProt Acc. No. Q9UNM7);
Aldo-keto reductase family 1 member B10 (SwissProt Acc. No. O60218);
Fructosamine-3-kinase (SwissProt Acc. No. Q9H479);
Peripherin (SwissProt Acc. No. P41219);
α-2-macroglobulin (SwissProt Acc. No. P01023);
Serpin C1 protein (SwissProt Acc. No. P01008);
Or apolipoprotein A IV (SwissProt Acc. No. P06727)
The method according to claim 19, wherein:   22. A method according to claim 21, characterized in that the change in protein concentration is detected by detecting a change in the level of autoantibodies relative thereto relative to the level of autoantibodies in the comparative sample.   23. A method according to any one of claims 19 to 22, characterized in that the effective body tissue is colorectal tissue.   The method according to any one of claims 19 to 22, characterized in that the effective body tissue is blood or a blood product such as serum or plasma.   25. A method according to any one of claims 19 to 24, characterized in that the treatment is surgery.   25. A method according to any one of claims 19 to 24, characterized in that the treatment is chemotherapy.   25. A method according to any one of claims 19 to 24, characterized in that the treatment is immunotherapy.   Recognizing the protein identified in claim 1, binding to the protein identified in claim 1, or of a partner substance having affinity for the protein identified in claim 1 for diagnostic or therapeutic purposes use.   Use according to claim 28, characterized in that the partner substance is an antibody.   30. Use according to claim 29, characterized in that the antibody is immobilized on a solid phase.   Use according to claim 30, characterized in that the antibody is immobilized on beads or as a chip.   Use according to claim 28, characterized in that the partner substance is an autoantibody.   21. A kit for carrying out the diagnostic method according to claim 1 or the therapeutic effect monitoring method according to claim 19, wherein the assay compatible format for interaction with a protein present in the diagnostic sample. Kit containing the partner substance.   34. Kit according to claim 33, characterized in that the assay compatibility format is a labeled format.   The kit according to claim 33 or 34, wherein the partner substance is an immobilized antibody.